The applicants overall approach to improvement in antifolate-based cancer treatment has been to seek detailed understanding in tumor and normal proliferative tissues that are the site of limiting toxicity of membrane transport, cellular pharmacokinetics and metabolic and enzymologic factors which govern antifolate action and determine therapeutic selectivity. Further advancement of this goal will be sought as follows. 1. Studies on folylpolyglutamyl synthetase. In light of our evidence documenting a role for FPGS in selective antitumor action and acquired resistance to 4- aminofolates; (a) Strategies will be utilized for obtaining additional tumor cell variants that overproduce FPGS and for deriving anti-FPGS monoclonal antibodies to be used to purify FPGS from these variants for biochemical and other studies described below; (b) Biochemical and genetic characterization of antifolate resistant tumor cell variants underproducing or overproducing FPGS will be carried out utilizing immunologic, cytogenetic and cell-cell hybridization methodology, (c) Relative content and regulation of this enzyme property in parental tumor cells and resistant variants with altered FPGS levels and in normal proliferative tissue will be examined in the context of proliferative state, growth arrest and differentiation. 2. Studies on folypolyglutamyl hydrolase (FPGH). As a continuation of earlier pharmacologic studies, we will attempt further purification of tumor-derived and cellular localization will be sought: 3. Metabolic turnover of 4-aminofolylpolyglutamates. We will continue our studies of lysosomal mediated hydrolysis of 4- aminofolylpolyglutamates focusing as well on the mechanism of lysomal internalization: 4. Studies on folate metabolism related to experimental therapeutic with antifolates and fluoropyrimidines. Work will be continued on the perturbations of reduced pools in tumor cells and normal proliferative tissues of antifolate treated mice. These will be extended to include studies of the metabolic disposition of Ca leucovorin and the role of 5,10-methenylTHF synthetase.